System and method for pre-programmed cold atmospheric plasma

ABSTRACT

To determine appropriate treatment doses of cold atmospheric plasma (CAP), the Canady Helios Cold Plasma Scalpel was tested across numerous cancer cell types including renal adenocarcinoma, colorectal carcinoma, pancreatic adenocarcinoma, ovarian adenocarcinoma, and esophageal adenocarcinoma. Various CAP doses were tested consisting of both high (3 L/min) and low (1 L/min) helium flow rates, several power settings, and a range of treatment times up to 5 minutes. The impact of cold plasma on the reduction of viability was consistently dose dependent, however the anti-cancer capability varied significantly between cell lines. While the lowest effective dose varied from cell line to cell line, in each case an 80-99% reduction in viability was achievable 48 hours after CAP treatment. Therefore, it is critical to select the appropriate CAP dose necessary for treating a specific cancer cell type.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of the filing date of U.S.Provisional Patent Application Ser. No. 62/797,855 filed by the presentinventors on Jan. 28, 2019.

The aforementioned provisional patent application is hereby incorporatedby reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

None.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to systems and methods for using coldatmospheric plasma to treat cancer.

Brief Description of the Related Art

Plasma medicine has qualified as a new scientific field after intenseresearch effort in low-temperature or cold atmospheric plasmaapplications. See, Laroussi M, Kong M, Morfill G, Stolz W, editors.Plasma medicine. Cambridge; 2012; Friedman A, Friedman G. Plasmamedicine. Hoboken: Wiley; 2013; and Keidar M, Beilis II. PlasmaEngineering: application in aerospace, nanotechnology andbionanotechnology. Oxford: Elsevier; 2013. It is known that coldatmospheric plasmas (CAP) produce various chemically reactive speciesincluding reactive oxygen species (ROS) and reactive nitrogen species(RNS). CAP is a cocktail containing ROS and RNS in combination withtransient electric fields, UV and charged species.

CAP has already been proven to be effective in wound healing, skindiseases, hospital hygiene, sterilization, antifungal treatments, dentalcare, and cosmetics targeted cell/tissue removal. See, Morfill G E, KongM G, Zimmermann J L. Focus on plasma medicine. Review. New J Phys. 2009;11:115011; Keidar M. Plasma for cancer treatment. Plasma Source SciTechnol. 2015; 24:033001; and Fridman G, Friedman G, Gutsol A, ShekhterA B, Vasilets V N, Fridman A. Applied plasma medicine. Plasma ProcessPolym. 2008; 5:503. One of the most recent applications of CAP is incancer therapy. See, Vandamme M, Robert E, Pesnel S, Barbosa E, DoziasS, Sobilo J, Lerondel S, Le Pape A, Pouvesle J M. Antitumor effect ofplasma treatment on U87 Glioma Xenografts: preliminary results. PlasmaProcess Polym. 2010; 7:264; Keidar M, Walk R, Shashurin A, Srinivasan P,Sandler A, Dasgupta S, Ravi R, Guerrero-Preston R, Trink B. Cold plasmaselectivity and the possibility of a paradigm shift in cancer therapy.Br J Cancer. 2011; 105:1295; and Vandamme M, Robert E, Lerondel S,Sarron V, Ries D, Dozias S, Sobilo J, Gosset D, Kieda C, Legrain B,Pouvesle J-M, Le Pape A. ROS implication in a new antitumor strategybased on non-thermal plasma. Int J Cancer. 2011; 130:2185. Multiplestudies have convincingly demonstrated that the CAP treatment leads toselective eradication of cancer cells in vitro and reduction of tumorsize in vivo. While most studies were done in vitro, some work was donein vivo. Recently, clinical cases of CAP application in cancer therapywere presented at the 2nd International Workshop on Plasma for CancerTherapy in Nagoya (Japan) and one of these studies involving 12 patientsafflicted with advanced squamous cell carcinoma of the head and neck hasbeen documented in a recent paper. See, Metelmann H R. What kind ofimpact is possible by plasma-jet in head and neck cancer?”, The 2ndInternational Workshop on Plasma for Cancer Treatment, Nagoya, Japan,March, 2015; Canady J. “Development and clinical application of hybridand cold atmospheric plasma combined with systemic chemotherapy andselective 3D conformal radiation therapy: A novel approach to thetreatment of peritoneal metastases from colorectal cancer.” The 2^(nd)International Workshop on Plasma for Cancer Treatment, Nagoya, Japan,March 2015; and Metelmann H R, Nedrelow D S, Seebauer C, Schuster M, vonWoedtke T, Weltmann K-D, Kindler S, Metelmann P H, Finkelstein S E, VonHoff D D, Podmelle F. Head and neck cancer treatment and physicalplasma. Clin Plasma Med. 2015; 3:17-23.

As a near-room temperature ionized gas, cold atmospheric plasma (CAP)has demonstrated its promising capability in cancer treatment by causingthe selective death of cancer cells in vitro. See, Yan D, Sherman J Hand Keidar M, “Cold atmospheric plasma, a novel promising anti-cancertreatment modality,” Oncotarget. 8 15977-15995 (2017); Keidar M, “Plasmafor cancer treatment,” Plasma Sources Sci. Technol. 24 33001 (2015);Hirst A M, Frame F M, Arya M, Maitland N J and O'Connell D, “Lowtemperature plasmas as emerging cancer therapeutics: the state of playand thoughts for the future,” Tumor Biol. 37 7021-7031 (2016). The CAPtreatment on several subcutaneous xenograft tumors and melanoma in micehas also demonstrated its potential clinical application. See, Keidar M,Walk R, Shashurin A, Srinivasan P, Sandler A, Dasgupta S, Ravi R,Guerrero-Preston R and Trink B, “Cold plasma selectivity and thepossibility of a paradigm shift in cancer therapy,” Br. J. Cancer. 1051295-301 (2011); Vandamme M, Robert E, Dozias S, Sobilo J, Lerondel S,Le Pape A and Pouvesle J-M, “Response of human glioma U87 xenografted onmice to non thermal plasma treatment,” Plasma Med. 1 27-43 (2011);Brulle L, Vandamme M, Ries D, Martel E, Robert E, Lerondel S, Trichet V,Richard S, Pouvesle J M and Le Pape A, “Effects of a Non thermal plasmatreatment alone or in combination with gemcitabine in a MIA PaCa2-lucorthotopic pancreatic carcinoma model,” PLoS One. 7 e52653 (2012); andChernets N, Kurpad D S, Alexeev V, Rodrigues D B and Freeman T A,“Reaction chemistry generated by nanosecond pulsed dielectric barrierdischarge treatment is responsible for the tumor eradication in the B16melanoma mouse model,” Plasma Process. Polym. 12 1400-1409 (2015).

Several different systems and methods for performing Cold AtmosphericPlasma (CAP) treatment have been disclosed. For example, U.S. Pat. No.10,213,614 discloses a two-electrode system for CAP treatement.

Another exemplary Cold Atmospheric Plasma system is disclosed in U.S.Pat. No.9,999,462. The disclosed system has two units, namely aConversion Unit (CU) and a Cold Plasma Probe (CPP). The Conversion Unitis connected to an electrosurgical generator (ESU) output and convertsthe ESU signal to a signal appropriate for performing cold atmosphericplasma procedures. The Cold Plasma Probe is connected to the ConversionUnit output. At the end of the Cold Plasma Probe cold plasma is producedand is thermally harmless to living tissue, i.e., it cannot cause burnsto the tissue. This cold plasma, however, is deadly for cancer cellswhile leaving normal cells unaffected. The disclosed Cold PlasmaConversion Unit is unique in that it utilizes a high voltage transformerto up-convert the voltage (1.5-50 kV), down-convert the frequency (<300kHz), and down-convert the power (<30 W) of the high-voltage output froman electrosurgical unit (U.S. Pat. No. 9,999,462).

Malignant solid tumors are characterized by high recurrence rates andlow 5-year survival rates. Stage IV renal adenocarcinoma presents anextremely low 5-year survival rate of 0-10% (see, Turhal, N., “Two casesof advanced renal cell cancer with prolonged survival of 8 and 12years,” Jpn J Clin Oncol 2002, 32, 152-153), while the recurrence ratemay be as high as 23%. See, Dabestani, S.; Beisland, C.; Stewart, G. D.;Bensalah, K.; Gudmundsson, E.; Lam, T. B.; Gietzmann, W.; Zakikhani, P.;Marconi, L.; Fernandez-Pello, S., et al., “Long-term outcomes offollow-up for initially localised clear cell renal cell carcinoma: Recurdatabase analysis,” Eur Urol Focus 2018. While the recurrence rate ofcolorectal carcinoma is similar to that of renal adenocarcinoma at 19.4%to 21.6%, the 5-year survival rate is significantly higher at 88.6% to89.4% [3]. See, Wille-Jorgensen, P.; Syk, I.; Smedh, K.; Laurberg, S.;Nielsen, D. T.; Petersen, S. H.; Renehan, A. G.; Horvath-Puho, E.;Pahlman, L.; Sorensen, H. T., et al., “Effect of more vs less frequentfollow-up testing on overall and colorectal cancer-specific mortality inpatients with stage ii or iii colorectal cancer: The colofol randomizedclinical trial,” JAMA 2018, 319, 2095-2103. Pancreatic ductaladenocarcinoma has an extremely low survival rate of 10% to 28% (Benzel,J.; Fendrich, V., “Chemoprevention and treatment of pancreatic cancer:Update and review of the literature,” Digestion 2018, 97, 275-287) afterone year due to a very high recurrence rate of 65.5%. See, Nakayama, Y.;Sugimoto, M.; Gotohda, N.; Konishi, M.; Takahashi, S., “Efficacy ofcompletion pancreatectomy for recurrence of adenocarcinoma in theremnant pancreas,” J Surg Res 2018, 221, 15-23. The resulting 5-yearsurvival rate is dismal at 6% in the United States and Europe. See,Conroy, T.; Desseigne, F.; Ychou, M.; Bouche, O.; Guimbaud, R.;Becouarn, Y.; Adenis, A.; Raoul, J. L.; Gourgou-Bourgade, S.; de laFouchardiere, C., et al., “Folfirinox versus gemcitabine for metastaticpancreatic cancer,” N Engl J Med 2011, 364, 1817-1825. Serous epithelialovarian carcinoma has a very low 5-year survival rate of 42% for stageIII and 26% for stage IV (see, Torre, L. A.; Trabert, B.; DeSantis, C.E.; Miller, K. D.; Samimi, G.; Runowicz, C. D.; Gaudet, M. M.; Jemal,A.; Siegel, R. L, “Ovarian cancer statistics 2018,” CA Cancer J Clin2018) with a 19% recurrence rate (Hou, M. M.; Chen, Y.; Wu, Y. K.; Xi,M. R., “Pathological characteristics and prognosis of 664 patients withepithelial ovarian cancer: A retrospective analysis,” Sichuan Da Xue XueBao Yi Xue Ban 2014, 45, 859-862, 875). Esophageal adenocarcinomarepresents a similarly low 5-year survival rate of 33% to 44% (see,Visser, E.; Edholm, D.; Smithers, B. M.; Thomson, I. G.; Burmeister, B.H.; Walpole, E. T.; Gotley, D. C.; Joubert, W. L.; Atkinson, V.; Mai,T., et al., “Neoadjuvant chemotherapy or chemoradiotherapy foradenocarcinoma of the esophagus,” J Surg Oncol 2018), depending on thetreatment used, and can result in a recurrence rate as high as 43.2%(see, Xi, M.; Yang, Y.; Zhang, L.; Yang, H.; Merrell, K. W.; Hallemeier,C. L.; Shen, R. K.; Haddock, M. G.; Hofstetter, W. L.; Maru, D. M., etal., “Multi-institutional analysis of recurrence and survival afterneoadjuvant chemoradiotherapy of esophageal cancer: Impact of histologyon recurrence patterns and outcomes,” Ann Surg 2018).

The unfortunately common recurrence rate of malignant solid tumorsrepresents a unique opportunity for cold atmospheric plasma (CAP)treatment. CAP can be used to treat the margins following tumor removal,and in doing so has the potential to remove residual cancer cells andprevent recurrence. An important step to make this a reality is todetermine the correct dose of CAP to significantly reduce tumor cellviability. It has been reported that various cell lines reactdifferently to CAP treatment [11-14]. See, Yan, D.; Talbot, A.;Nourmohammadi, N.; Cheng, X.; Canady, J.; Sherman, J.; Keidar, M.,“Principles of using cold atmospheric plasma stimulated media for cancertreatment,” Sci Rep 2015, 5, 18339; Naciri, M.; Dowling, D.; Al-Rubeai,M., “Differential sensitivity of mammalian cell lines to non-thermalatmospheric plasma,” Plasma Processes and Polymers 2014, 11, 391-400;and Ma, Y.; Ha, C. S.; Hwang, S. W.; Lee, H. J.; Kim, G. C.; Lee, K. W.;Song, K., “Non-thermal atmospheric pressure plasma preferentiallyinduces apoptosis in p53-mutated cancer cells by activating rosstress-response pathways,” PLoS One 2014, 9, e91947. Dayun Yan, JonathanH. Sherman, Jerome Canady, Barry Trink, Michael Keidar, “The cellularros-scavenging function, a key factor determining the specificvulnerability of cancer cells to cold atmospheric plasma in vitro,”arXiv.org; arXiv:1711.09015: 2017; Vol. arXiv:1711.09015. Yan et al.studied the reactive species consumption speed of glioblastoma U87 andbreast cancers MDA-MB-231 and MCF-7, and discovered that the cancercells that could absorb or eliminate the effective species in the mediafaster (glioblastoma) would be more resistant to plasma-activated mediumthan both breast cancers. Their results also demonstrate a wide range ofeffects on cell viability depending on cell type and treatment timeused. Naciri et al. also reported that plasma sensitivity closelycorrelates with proliferation rates by measuring ATP levels of 3 cancercell types including Chinese hamster ovary cells, osteoblast, and colonadenocarcinoma. By testing 8 cancerous cell lines, Ma et al. claimedthat p53-deficient cancer cells are more sensitive to CAP treatment dueto the lack of p53-dependent cell cycle delay at G1. Therefore, thecombinations of power settings and treatment times are critical for thecorrect dosage of cell line-dependent CAP treatment establishment.

The mechanism of anti-cancer capacity of CAP has been increasinglyunderstood. Several theories have been proposed, including decrease ofcell adhesion, interruption of cell cycle, induction of apoptosis andDNA fragmentation. For decrease of cell adhesion, see Lee, H. J.; Shon,C. H.; Kim, Y. S.; Kim, S.; Kim, G. C.; Kong, M. G., “Degradation ofadhesion molecules of g361 melanoma cells by a non-thermal atmosphericpressure microplasma,” New Journal of Physics 2009, 11 and Schmidt, A.;Bekeschus, S.; von Woedtke, T.; Hasse, S., “Cell migration and adhesionof a human melanoma cell line is decreased by cold plasma treatment,”Clinical Plasma Medicine 2015, 3, 24-31. For interruption of cell cycle,see, Shi, X.-M.; Zhang, G.-J.; Chang, Z.-S.; Wu, X.-L.; Liao, W.-L.; Li,N., “Viability reduction of melanoma cells by plasma jet via inducingg1/s and g2/m cell cycle arrest and cell apoptosis,” IEEE Transactionson Plasma Science 2014, 42, 1640-1647; Chang, J. W.; Kang, S. U.; Shin,Y. S.; Kim, K. I.; Seo, S. J.; Yang, S. S.; Lee, J. S.; Moon, E.; Baek,S. J.; Lee, K., et al., “Non-thermal atmospheric pressure plasma inducesapoptosis in oral cavity squamous cell carcinoma: Involvement ofDNA-damage-triggering sub-g(1) arrest via the atm/p53 pathway,” ArchBiochem Biophys 2014, 545, 133-140, and Gherardi, M.; Turrini, E.;Laurita, R.; De Gianni, E.; Ferruzzi, L.; Liguori, A.; Stancampiano, A.;Colombo, V.; Fimognari, C., “Atmospheric non-equilibrium plasma promotescell death and cell-cycle arrest in a lymphoma cell line,” PlasmaProcesses and Polymers 2015, 12, 1354-1363

For induction of apoptosis, see Adachi, T.; Tanaka, H.; Nonomura, S.;Hara, H.; Kondo, S.; Hori, M., “Plasma-activated medium induces a549cell injury via a spiral apoptotic cascade involving themitochondrial-nuclear network,” Free Radic Biol Med 2015, 79, 28-44;Ahn, H. J.; Kim, K. I.; Kim, G.; Moon, E.; Yang, S. S.; Lee, J. S.,“Atmospheric-pressure plasma jet induces apoptosis involvingmitochondria via generation of free radicals,” PLoS One 2011, 6, e28154;and Kim, S. J.; Chung, T. H.; Bae, S. H.; Leem, S. H., “Induction ofapoptosis in human breast cancer cells by a pulsed atmospheric pressureplasma jet,” Applied Physics Letters 2010, 97; and Keidar, M.; Walk, R.;Shashurin, A.; Srinivasan, P.; Sandler, A.; Dasgupta, S.; Ravi, R.;Guerrero-Preston, R.; Trink, B., “Cold plasma selectivity and thepossibility of a paradigm shift in cancer therapy,” Br J Cancer 2011,105, 1295-1301.

For DNA fragmentation, see Nuccitelli, R.; Chen, X.; Pakhomov, A. G.;Baldwin, W. H.; Sheikh, S.; Pomicter, J. L.; Ren, W.; Osgood, C.;Swanson, R. J.; Kolb, J. F., et al., “A new pulsed electric fieldtherapy for melanoma disrupts the tumor's blood supply and causescomplete remission without recurrence,” Int J Cancer 2009, 125, 438-445.However, it is not yet clear whether CAP generated reactive species arecrucial for apoptosis and its associated DNA strand break or whetherplasma-induced direct DNA damage provokes cell cycle checkpointsignaling that leads to apoptosis. Chung, W. H., “Mechanisms of a novelanticancer therapeutic strategy involving atmospheric pressureplasma-mediated apoptosis and DNA strand break formation,” Arch PharmRes 2016, 39, 1-9.

SUMMARY OF THE INVENTION

In a preferred embodiment, the present invention is a pre-programmedsystem for performing cold atmospheric plasma treatment on targettissue. The system has an RF energy source, a low frequency conversionunit, a gas control device, a processor configured to control the RFenergy module, the LF conversion module and the gas control module, adisplay, graphical user interface displayed on the display forcontrolling cold atmospheric plasma procedures, a data storage unit ormemory; and a database stored in the data storage unit or memory, thedatabase comprising cell lines identifying data and cold atmosphericplasma settings associated with the cell line identifying data. Thegraphical user interface has input means for a surgeon to enter cellline identifier data and in response to the entry of cell lineidentifier data the electrosurgical generator automatically accesses thedatabase in the data storage or memory and adopts cold atmosphericplasma settings associated with the entered cell line identifier data inthe database and displays the adopted cold atmospheric plasma settingson the display. The RF energy source may be a monopolar electrosurgicalgenerator, gas control module and LF conversion unit may be separatedevices or may be within an integrated cold atmospheric plasma generatoror a variation in which the RF energy and conversion unit are in anintegrated unit while the gas control module is in a separate device. Ina preferred embodiment, the processor is in the integrated coldatmospheric plasma generator. The data storage unit or memory may bewithin the integrated cold atmospheric plasma generator or may beexternal thereof. The display comprises a touchscreen, for example, on atablet computer. The data storage or memory may be a memory in theprocessor. The cold atmospheric plasma settings may comprise at leasttwo of power, flow rate and time.

In another preferred embodiment, the present invention is a method forapplying cold atmospheric plasma treatment on target tissue. The methodcomprises the steps of selecting through a graphical user interface on adisplay a particular cancer cell line associated with the target tissue,retrieving in response to the selecting, with a computing deviceconnected to the display, settings data from a database of cell linedata and associated settings data in a storage, applying, with thecomputing device, the retrieved settings data to a cold atmosphericplasma system, and treating cancer tissue with cold atmospheric plasmaat the retrieved settings. The method may further comprise displayingthe retrieved settings on a display.

Still other aspects, features, and advantages of the present inventionare readily apparent from the following detailed description, simply byillustrating a preferable embodiments and implementations. The presentinvention is also capable of other and different embodiments and itsseveral details can be modified in various obvious respects, all withoutdeparting from the spirit and scope of the present invention.Accordingly, the drawings and descriptions are to be regarded asillustrative in nature, and not as restrictive. Additional objects andadvantages of the invention will be set forth in part in the descriptionwhich follows and in part will be obvious from the description or may belearned by practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention and theadvantages thereof, reference is now made to the following descriptionand the accompanying drawings, in which:

FIG. 1A is a perspective view of a preferred embodiment of agas-enhanced electrosurgical generator.

FIG. 1B is a front view of a preferred embodiment of a gas-enhancedelectrosurgical generator.

FIG. 1C is a rear view of a preferred embodiment of a gas-enhancedelectrosurgical generator.

FIG. 1D is a left side view of a preferred embodiment of a gas-enhancedelectrosurgical generator.

FIG. 1E is a right view of a preferred embodiment of a gas-enhancedelectrosurgical generator.

FIG. 1F is a top view of a preferred embodiment of a gas-enhancedelectrosurgical generator.

FIG. 1G is a bottom view of a preferred embodiment of a gas-enhancedelectrosurgical generator.

FIG. 2A is a block diagram of a preferred embodiment of a gas-enhancedelectrosurgical generator having a pressure control system in accordancewith the present invention configured to perform an argon-enhancedelectrosurgical procedure.

FIG. 2B is a block diagram of a preferred embodiment of a gas-enhancedelectrosurgical generator having a pressure control system in accordancewith the present invention configured to perform a cold atmosphericplasma procedure.

FIG. 2C is a diagram of a trocar for the embodiment of FIG. 2A inaccordance with the present invention.

FIG. 2D is a block diagram of an alternate preferred embodiment ofpressure control system of a gas-enhanced electrosurgical generatorhaving a pressure control system in accordance with the presentinvention configured to perform an argon-enhanced electrosurgicalprocedure.

FIG. 3A is a schematic flow diagram illustrating the gas flow throughthe module and the method by which the module controls the gas flow inaccordance with a preferred embodiment of the present invention.

FIG. 3B is a schematic flow diagram illustrating the gas flow through analternate embodiment of the module and the method by which the modulecontrols the gas flow in accordance with a preferred embodiment of thepresent invention.

FIG. 4 is a diagram of a graphical user interface in accordance with apreferred embodiment of the present invention.

FIGS. 5A and 5B illustrate a reduction of viability of 769-P followingCAP treatment. CAP treatment of 769-P significantly reduces viability atall doses tested.

FIGS. 6A and 6B illustrate reduction of viability of HCT-116 followingCAP treatment. CAP treatment of HCT-116 significantly reduces viabilityat all doses tested.

FIGS. 7A and 7B illustrates reduction of viability of SK-OV-3 followingCAP treatment. CAP treatment of SK-OV-3 significantly reduces viabilityat nearly all doses tested using 3 L/min (FIG. 8A) and at all dosesusing 1 L/min flow rate (FIG. 8B).

FIGS. 8A and 8B illustrates reduction of viability of BxPC-3 followingCAP treatment. CAP treatment of BxPC-3 significantly reduces viabilityat nearly all doses tested.

FIGS. 9A and 9B illustrates reduction of viability of OE33 following CAPtreatment. CAP treatment of OE33 significantly reduces viability at alldoses tested.

FIGS. 10A and 10B illustrate an experimental setup for solid tumortreatment with cold atmospheric plasma. FIG. 10A illustrates cellscultured in a 12-well plate, treated using a Canady Helios PlasmaScalpel at 3 L/min. FIG. 10B illustrates an electrosurgical generatorconnected to a Cold Plasma Conversion Unit.

FIG. 11 is a flow chart of a method for performing CAP treatment inaccordance with a preferred embodiment of the present invention.

FIG. 12 is a diagram of an exemplary CAP database in accordance with apreferred embodiment of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As shown in the experiments discussed below, various cancer cell linecan be tested to provide a rough prediction of which cells lines aresusceptible to treatment with CAP and further, the various cancer celllines can be tested at varying settings or dosages of the CAP treatmenton provide an estimate of which CAP treatment settings or dosages willprovide the greatest effect on particular cancer cell lines. In apreferred embodiment of the present invention, the results of suchtesting are used to generate a database of cancer cell lines withassociated predicted optimum settings or dosage data and optionallyeffectiveness data. This database can be stored in memory or otherstorage in a CAP capable electrosurgical system or can be in an externalstorage, for example, accessible through a server or cloud computingsystem, that can be accessed by a CAP capable electrosurgical system.The database effectively pre-programs the system to treat a particularcancer cell line with CAP at particular settings. The CAP capableelectrosurgical system may have a graphical user interface that allows auser to enter an identifier for a particular cancer cell line into theuser interface and thereby have the CAP-enabled electrosurgical systemautomatically select the predicted optimum settings or dosage for thatparticular cancer cell line. The user can then perform a CAP treatmentof target cancer cells at those predicted optimum settings.

Thus, as shown in FIG. 11 , a method can be performed in which adatabase of cancer cell lines and associated CAP treatment data isgenerated and stored (step 1110). Additional cancer cell line data andassociate settings or dosage data can be added to the database as newcell lines are tested and new data is developed. A user performing a CAPtreatment determines the particular cancer cell line to be treated (step1120) and then enters an identifier associated with a particular cancercell line into a graphical user interface on a CAP capableelectrosurgical system (step 1130). The CAP capable electrosurgicalsystem then accesses the stored database to retrieve CAP setting ordosages associated with the ID entered into the graphical user interface(step 1140). The phrase “enter an identifier” used herein can mean anydata entry or selection by the user that provides the graphical userinterface with sufficient information to retrieve data from the databasefor a particular cell line. This could be selection from a list or menu,entry of an identifier through a physical or virtual keyboard associatedwith the system, scanning of a bar code, or any other means. Further,the graphical user interface and associated display do not need tophysically be in the CAP capable generator but instead may be onexternal devices such as a tablet computing device that is incommunication with the CAP enabled electrosurgical generator. Theretrieved CAP settings are then applied to the CAP system (step 1150).The user then can treat the target tissue with CAP at the preferredsettings (step 1160).

A preferred embodiment of a CAP enable generator is described withreference to the drawings. A gas-enhanced electrosurgical generator 100in accordance with a preferred embodiment of the present invention isshown in FIGS. 1A-1G. The gas-enhanced generator has a housing 110 madeof a sturdy material such as plastic or metal similar to materials usedfor housings of conventional electrosurgical generators. The housing 110has a removable cover 114. The housing 110 and cover 114 have means,such as screws 119, tongue and groove, or other structure for removablysecuring the cover to the housing. The cover 114 may comprise just thetop of the housing or multiple sides, such as the top, right side andleft side, of the housing 110. The housing 110 may have a plurality offeet or legs 140 attached to the bottom of the housing. The bottom 116of the housing 110 may have a plurality of vents 118 for venting fromthe interior of the gas-enhanced generator.

On the face 112 of the housing 114 there is a touch-screen display 120and a plurality of connectors 132, 134 for connecting variousaccessories to the generator, such as an argon plasma probe, a hybridplasma probe, a cold atmospheric plasma probe, or any otherelectrosurgical attachment. There is a gas connector 136 for connecting,for example, a CO₂ supply for insufflating an abdomen. The face 112 ofthe housing 110 is at an angle other than 90 degrees with respect to thetop and bottom of the housing 110 to provide for easier viewing and useof the touch screen display 120 by a user.

One or more of the gas control modules may be mounting within agas-enhanced electrosurgical generator 100. A gas pressure controlsystem 200 for controlling a plurality of gas control modules 220, 230,240 within a gas-enhanced electrosurgical generator is described withreference to FIGS. 2A-2D. A plurality of gas supplies 222, 232, 242 areconnected to the gas pressure control system 200, and more specifically,to the respective gas control modules 220, 230, 240 within the gaspressure control system 200. The gas pressure control system 200 has apower supply 202 for supplying power to the various components of thesystem. A CPU 210 controls the gas pressure control modules 220, 230,240 in accordance with settings or instructions entered into the systemthrough a graphical user interface on the display 120. The system isshown with gas control modules for CO₂, argon and helium, but the systemis not limited to those particular gases. In the embodiment shown inFIGS. 2A-2D, the CO₂ is shown as the gas used to insufflate an abdomen(or other area of a patient). The gas pressure control system 200 has a3-way proportional valve connected to the gas control module 220. WhileFIG. 2A shows the 3-way proportional valve connected only to the CO2control module 220, the 3-way proportional valves could be connected toa different gas control module 230 or 240. The gas pressure controlsystem 200 further has an HF power module 250 for supplying highfrequency electrical energy for various types of electrosurgicalprocedures. The HF power module contains conventional electronics suchas are known for provide HF power in electrosurgical generators.Exemplary systems include, but are not limited to, those disclosed inU.S. Pat. No. 4,040,426 and U.S. Pat. No. 4,781,175. The system furthercould have a converter unit for converting the HF power to a lowerfrequency, such as may be used for cold atmospheric plasma and isdescribed in U.S. Patent Application Publication No. 2015/0342663.

The outlet port of gas control module 220 is connected to a connector136 on the generator housing. While connector 136 and the otherconnectors are shown on the front face of the housing 110, they could beelsewhere on the housing. The outlet ports of gas control modules 230,240 each are connected to tubing or other channel to a connector 132. Aconnector 152 connects to connector 136 and is as tubing that runs toand connects to tubing 292. The tubing 292 is connected to a pressurecontrol valve or stopcock 280 and extends into the trocar. The pressurecontrol valve 280 is used to control pressure within the patient. Thegas pressure control system further has a pressure sensor 282 connectedto the tubing 292 to sense pressure in the tubing 292 and a pressuresensor 284 for sensing pressure in the pressure control valve 280. Asshown in FIG. 2C, the tubing 292 is actually tube within a tube suchthat gas supplied from the generator travels to the trocar and patientthrough tube 296 and gas is released out of the patient through tube294.

As shown in FIG. 2A the connector 132 to which control module 230 isconnected has a gas-enhanced electrosurgical instrument 160 having aconnector 162 connected to in. In FIG. 2A, gas control module 230controls flow of argon gas, so the instrument 160 is an argongas-enhanced electrosurgical tool such as an argon plasma probe such asis disclosed in U.S. Pat. No. 5,720,745, a hybrid plasma cut accessorysuch as is disclosed in U.S. Patent Application Publication No.2017/0312003 or U.S. Patent Application Publication No. 2013/0296846, ora monopolar sealer such as is disclosed in U.S. Patent ApplicationPublication No. 2016/0235462. Other types of argon surgical devicessimilarly can be used. As shown in FIG. 2B the connector 132 to whichcontrol module 240 is connected has a gas-enhanced electrosurgicalinstrument 170 having a connector 172 connected to in. In FIG. 2B, gascontrol module 240 controls flow of helium gas, so the instrument 170is, for example, a cold atmospheric plasma attachment such as isdisclosed in U.S. Patent Application Publication No. 2016/0095644.

The system provides for control of intraabdominal pressure in a patient.The pressure control valve 280 has a chamber within it. The pressure inthat chamber is measured by pressure sensor 284. CO₂ is supplied to thechamber within pressure control valve 280 from gas control module 220via 3-way proportional valve 260. Pressure in that chamber within thepressure control valve 280 also may be released via 3-way proportionalvalve 260. In this manner, the system can use the pressure sensor 284and the 3-way proportional valve to achieve a desired pressure (setthrough a user interface) in the chamber within the pressure controlvalve 280. The pressure sensor 282 senses the pressure in the tubing 294(and hence the intraabdominal pressure). The pressure control valve 280then releases pressure through its exhaust to synchronize theintraabdominal pressure read by sensor 282 with the pressure in thechamber within the pressure control valve as read by pressure sensor284. The readings from sensors 282, 284 can be provided to CPU 210,which in turn can control flow of CO₂ and one of argon and helium,depending on the procedure being performed, to achieve a stable desiredintraabdominal pressure.

An alternative embodiment of the gas pressure control system is shown inFIG. 2D. This this system the automatic stopcock or pressure controlvalve 280 has been replaced by a manual relief valve 280 a that iscontrolled or operated by the surgeon using the system.

A gas control module 300 in accordance with the present invention isdesigned for gas-enhanced electrosurgical systems. Conventionally,gas-enhanced electrosurgical systems have an electrosurgical generatorand a gas control unit that have separate housings. The conventional gascontrol unit typically controls only a single gas such as argon, CO₂ orhelium. The present invention is a gas control module 300 that may beused in a gas control unit or in a combined unit functioning both as anelectrosurgical generator and as a gas control unit. Further, aplurality of gas control modules in accordance with the presentinvention may be combined in a single gas control unit or combinationgenerator/gas control unit to provide control of multiple gases andprovide control for multiple types of gas-enhanced surgery such as argongas coagulation, hybrid plasma electrosurgical systems and coldatmospheric plasma systems.

FIG. 3A is a schematic flow diagram illustrating the gas flow throughthe gas control module 300 and the method by which the module 300controls the gas flow in accordance with a preferred embodiment of thepresent invention. As shown in FIG. 3A, the gas enters the gas controlmodule at an inlet port (IN) 301 and proceeds to first solenoid valve(SV1) 310, which is an on/off valve. In an exemplary embodiment, the gasenters the gas module at a pressure of 75 psi. The gas then proceeds toa first pressure sensor (P1) 320, to a first pressure regulator (R1)330. In an exemplary embodiment, the first pressure regulator (R1) 330reduces the pressor of the gas from 75 psi to 18 psi. After the pressureregulator (R1) 330, the gas proceeds to flow sensor (FS1) 340, whichsense the flow rate of the gas. Next, the gas proceeds to proportionalvalve (PV1) 350, which permits adjustment of a percentage of the openingin the valve. The gas then proceeds to a second flow sensor (FS2) 360,which senses the flow rate of the gas. This second flow sensor (FS2) 360provides redundancy and thus provides greater safety and accuracy in thesystem. Next the gas proceeds to a second solenoid valve (SV2) 370,which is a three-way valve that provides for a vent function that canallow gas to exit the module through a vent 372. The gas then proceedsto a second pressure sensor (P2) 380, which provides a redundantpressure sensing function that against produces greater safety andaccuracy of the system. Finally, the gas proceeds to a third solenoidvalve (SV3) 390, which is a two-way on/off valve that is normally closedand is the final output valve in the module. The gas exits the module atand output port (OUT) 399, which is connected to tubing or other channelthat provides a passageway for the gas to flow to an accessory connectedto the electrosurgical unit.

FIG. 3B is a schematic flow diagram of an alternate embodiment of a gascontrol module illustrating the gas flow through the gas control module300 a and the method by which the module 300 a controls the gas flow inaccordance with a preferred embodiment of the present invention. Asshown in FIG. 3B, the gas enters the gas control module at an inlet port301 a and proceeds to a first pressure regulator (R1) 330 a. In anexemplary embodiment, the first pressure regulator (R1) 330 a reducesthe pressor of the gas from about 50-100 psi to 15-25 psi. After thepressure regulator (R1) 330 a, the gas proceeds to a first pressuresensor (P1) 320 a and then to a first solenoid valve (SV1) 310 a, whichis an on/off valve. Next, the gas proceeds to proportional valve (PV1)350 a, which permits adjustment of a percentage of the opening in thevalve. Next, the gas proceeds to flow sensor (FS1) 340 a, which sensethe flow rate of the gas. ext the gas proceeds to a second solenoidvalve (SV2) 370 a, which is a three-way valve that provides for a ventfunction that can allow gas to exit the module through a vent 372 a. Thegas then proceeds to a second flow sensor (FS2 ) 360 a, which senses theflow rate of the gas. This second flow sensor (FS2) 360 a providesredundancy and thus provides greater safety and accuracy in the system.The gas then proceeds to a second pressure sensor (P2) 380 a, whichprovides a redundant pressure sensing function that against producesgreater safety and accuracy of the system. The gas exits the module atand output port 399 a, which is connected to tubing or other channelthat provides a passageway for the gas to flow to an accessory connectedto the electrosurgical unit.

The various valves and sensors in either embodiment of the module areelectrically connected to a main PCB Board through a connector 490. ThePCB connector 490 is connected to a PCB Board that has a microcontroller(such as CPU 210 in the embodiment shown in FIG. 2A). As previouslynoted, a plurality of gas modules can be in a single gas control unit orsingle electrosurgical generator to provide control of multiplediffering gases. The plurality of gas control modules further may beconnected to the same PCB Board, thus providing common control of themodules.

As shown in FIG. 4 , the generator further may have graphical userinterface 400 for controlling the components of the system using thetouch screen display 120. The graphical user interface 400 for example,may control robotics 411, argon-monopolar cut/coag 412, hybrid plasmacut 413, cold atmospheric plasma 414, bipolar 415, plasma sealer 416,hemo dynamics 417 or voice activation 418. The graphical user interfacefurther may be used with fluorescence-guided surgery 402. For example,J. Elliott, et al., “Review of fluorescence guided surgery visualizationand overlay techniques,” BIOMEDICAL OPTICS EXPRESS 3765 (2015), outlinesfive practical suggestions for display orientation, color map,transparency/alpha function, dynamic range compression and colorperception check. Another example of a discussion of fluorescence-guidedsurgery is K. Tipirneni, et al., “Oncologic Procedures Amenable toFluorescence-guided Surgery,” Annals of Surgery, Vo. 266, No. 1, Jul.2017). The graphical user interface (GUI) further may be used withguided imaging such as CT, MRI or ultrasound. The graphical userinterface may communicate with RFID 420 (such as may be found in variouselectrosurgical attachments) and may collect and store usage data in astorage medium 430. The graphical user interface 400 communicates withFPGA 440, which may control irrigation pump 452, insufflator 454, PFC462, full bridge 464 for adjusting the power output, fly back 466 forregulating the power (DC to AC) and a foot pedal 470. The GUI 400further communicates with a database of cancer cell line data withassociated predicted CAP settings or dosages via the CPU 210. Thedatabases storage may be internal memory or other internal storage 211or external storage 212 as shown in FIGS. 2A and 2B. The data storage430 in FIG. 4 may be in one or both of these memories or storages 211 or212.

Experiments for Determining Optimum CAP Settings

To develop the present invention, a Cold Atmospheric Plasma (CAP) devicewas tested on a wide range of cancer cell lines with differentcombinations of power settings, treatment times, and gas flow rate. Inthis way, optimal dosages for each cancer type were determined andrecorded. The following test procedures were sued. To determine theeffective plasma dose required to significantly reduce cell viabilitytwo flowrates were chosen; 1 L/min and 3 L/min of helium. Based oninitial testing 1-5 minutes with 40-80 power, and 30-120 sec with 20-40power, were chosen as an effective range for 3 L/min and 1 L/min,respectively. The power settings of 20P, 40P, 60P, and 80P used in thisstudy are 5 W, 8 W, 11 W, and 15.7 W at 3 L/min. At 1 L/min of 20P and40P, the power are 5 W and 6 W respectively. The detailed powermeasurement of our CAP device was conducted and reported in anotherpaper which is currently under review.

MTT assays were used to determine the dose of CAP needed tosignificantly reduce cell viability. MTT assays were performed on CAPtreated cancer cell lines 48 hours post treatment. The viability of thetreated cells was normalized to an untreated (control) group.

Results

Reduction of Cell Viability by CAP in Malignant Solid Tumor Cell Lines

Viability of 769-P renal adenocarcinoma cells was dose-dependent andsignificantly reduced at all time and power combinations tested (FIGS.5A and 5B). Helium flow alone (0W) did not significantly impact cellviability (FIG. 5A). At the highest doses using 3 L/min viability wasreduced to 4.9% (P <0.001) while viability at 20P 120 seconds was <1% (P<0.001) for 1 L/min. Increasing the power to 40P did not result in afurther reduction (P=0.65) (FIG. 5B). CAP was equally as effective inreducing viability in HCT-116 colorectal carcinoma cells. At 3 L/minviability was 76% at 40P 1 minute (P <0.01) and this decreased to 3.6%at the highest dose of 80P 5 minutes (P<0.001) (FIG. 6A). The decreasedin viability when using 1 L/min required a lower dose. Initially, at 20P30 seconds and 60 seconds, viability was reduced to 27% (P<0.001) and21% (P<0.01), respectively (FIG. 6B). However, beginning at 20P 90seconds viability was reduced to 5.0% (P<0.001) which only slightlydecreased with a higher dose, the highest dose resulting in 2.6%viability (P<0.001). CAP also had a clear dose-dependent effect in thereduction of viability in ovarian adenocarcinoma cell line; SK-OV-3. Atthe lowest dose, using 3 L/min, viability was only reduced to 87%(P<0.001) which decreased to 17% (P<0.001) at 80P for 5 minutes (FIG.7A). Similar results were found with the lower flow rate (FIG. 7B). 20Pfor 30 seconds resulted in 77% viability (P<0.001) which decreased to 4%viability (P<0.0001) at 40P for 120 seconds. BxPC-3 (pancreaticadenocarcinoma) required a higher dose to effectively reduce viability(FIG. 8A). At 60P and 5 minutes and 80P for 5 minutes viability wasreduced to 14% (P<0.0001) and 4% (P<0.0001), respectively. The low flowrate treatment showed a similar pattern requiring a higher dose toreduce viability. At 40P for 90 seconds and 40P for 120 seconds theviability was reduced to 5% (P<0.0001) and 1% (P<0.0001), respectively(FIG. 8B). Esophageal adenocarcinoma cell line, OE33, also required ahigher dose to decrease viability below 20%. Using the high flow rate at80P for 5 minutes viability was reduced to 16% (P<0.001) (FIG. 9A). At 1L/min an 40P for 120 seconds viability was reduced to 15% (P<0.0001)(FIG. 9B). Taken together, this data demonstrates that CAP reduced cellviability in a time- and power-dependent manner in all cell linestested.

Discussion

This present work represents the first effort to describe thedose-dependent reduction of viability, as a combination of treatmenttime and power settings, on multiple malignant solid tumor cell linesusing a CAP system. This CAP does not induce any damage on normaltissue. CAP treatment consistently resulted in a dose-dependentreduction in viability on all solid tumor cell lines tested. While thelowest effective dose varied from cell line to cell line, in each casean 80-99% reduction in viability was achievable 48 hours after CAPtreatment. 769-P and HCT-116 required a lower dose of plasma whileSK-OV-3, BxPC-3, and OE33 required a relatively higher dose. In all celllines tested, helium treatment alone (OP) showed no decrease inviability, indicating that the observed effects are due to CAP. While inseveral of the cell lines 1 L/min flow rate resulted in a lowerviability at a lower dose, this cannot be directly compared with the 3L/min results. This is because the beam length, media volume, and cellnumber are different between these two assays. Xu and Dai et al. havesuggested a formula to compare plasma dose among treatment conditionswithin one cell type and this may be necessary to compare future result.See, Xu, X.; Dai, X.; Xiang, L.; Cai, D.; Xiao, S.; Ostrikov, K.,“Quantitative assessment of cold atmospheric plasma anti-cancer efficacyin triple-negative breast cancers,” Plasma Processes and Polymers 2018.

Ma et al. demonstrated that the effectiveness of non-thermal plasmatreatment was partially dependent on p53 expression [13]. The viabilityof cancer cells lacking p53 was significantly reduced by non-thermalplasma treatment while p53⁺ cells were less affected. It is thought thatthis is due to the role of p53 in protecting the cell from reactiveoxygen species. See, Sablina, A. A.; Budanov, A. V.; Ilyinskaya, G. V.;Agapova, L. S.; Kravchenko, J. E.; Chumakov, P. M., “The antioxidantfunction of the p53 tumor suppressor,” Nat Med 2005, 11, 1306-1313.However, based on established literature, the cell lines tested here,except for SK-OV-3, have been shown to be positive for p53 (Table 1,below).

For 769-P see Wang, J.; Zhang, P.; Zhong, J.; Tan, M.; Ge, J.; Tao, L.;Li, Y. Zhu, Y.; Wu, L.; Qiu, J., et al., “The platelet isoform ofphosphofructokinase contributes to metabolic reprogramming and maintainscell proliferation in clear cell renal cell carcinoma,” Oncotarget 2016,7, 27142-27157; Miyazaki, J.; Ito, K.; Fujita, T.; Matsuzaki, Y.; Asano,T.; Hayakawa, M.; Asano, T.; Kawakami, Y., “Progression of human renalcell carcinoma via inhibition of rhoa-rock axis by parg1,” Transl Oncol2017, 10, 142-152; Mu, W.; Hu, C.; Zhang, H.; Qu, Z.; Cen, J.; Qiu, Z.;Li, C.; Ren, H.; Li, Y.; He, X., et al., “Mir-27b synergizes withanticancer drugs via p53 activation and cyplb1 suppression,” Cell Res2015, 25, 477-495; and Bamford, S.; Dawson, E.; Forbes, S.; Clements,J.; Pettett, R.; Dogan, A.; Flanagan, A.; Teague, J.; Futreal, P. A.;Stratton, M.R., et al., “The cosmic (catalogue of somatic mutations incancer) database and website,” Br J Cancer 2004, 91, 355-358.

For HCT-116 see Ma, Y.; Ha, C. S.; Hwang, S. W.; Lee, H. J.; Kim, G. C.;Lee, K. W.; Song, K., “Non-thermal atmospheric pressure plasmapreferentially induces apoptosis in p53-mutated cancer cells byactivating ros stress-response pathways,” PLoS One 2014, 9, e91947 andO'Connor, P. M.; Jackman, J.; Bae, I.; Myers, T. G.; Fan, S.; Mutoh, M.;Scudiero, D. A.; Monks, A.; Sausville, E. A.; Weinstein, J. N., et al.,“Characterization of the p53 tumor suppressor pathway in cell lines ofthe national cancer institute anticancer drug screen and correlationswith the growth-inhibitory potency of 123 anticancer agents,” Cancer Res1997, 57, 4285-4300.

For SK-OV-3, see Bamford, S.; Dawson, E.; Forbes, S.; Clements, J.;Pettett, R.; Dogan, A.; Flanagan, A.; Teague, J.; Futreal, P. A.;Stratton, M. R., et al., “The cosmic (catalogue of somatic mutations incancer) database and website,” Br J Cancer 2004, 91, 355-358; O'Connor,P. M.; Jackman, J.; Bae, I.; Myers, T. G.; Fan, S.; Mutoh, M.; Scudiero,D. A.; Monks, A.; Sausville, E. A.; Weinstein, J. N., et al.,“Characterization of the p53 tumor suppressor pathway in cell lines ofthe national cancer institute anticancer drug screen and correlationswith the growth-inhibitory potency of 123 anticancer agents,” Cancer Res1997, 57, 4285-4300; Antoun, S., Atallah, D., Tahtouh, R., Alaaeddine,N., Moubarak, M., Khaddage, A., Ayoub, E. N., Chahine, G., and Hilal,G., “Different tp53 mutants in p53 overexpressed epithelial ovariancarcinoma can be associated both with altered and unaltered glycolyticand apoptotic profiles,” Cancer Cell Int 2018, 18, 14; and Yaginuma, Y.;Westphal, H., “Abnormal structure and expression of the p53 gene inhuman ovarian carcinoma cell lines,” Cancer Res 1992, 52, 4196-4199

For BxPC-3, see Chen, D.; Niu, M.; Jiao, X.; Zhang, K.; Liang, J.;Zhang, D., “Inhibition of akt2 enhances sensitivity to gemcitabine viaregulating puma and nf-kappab signaling pathway in human pancreaticductal adenocarcinoma,” Int J Mol Sci 2012, 13, 1186-1208; Wang, F.; Li,H.; Yan, X. G.; Zhou, Z. W.; Yi, Z. G.; He, Z. X.; Pan, S. T.; Yang, Y.X.; Wang, Z. Z.; Zhang, X., et al., “Alisertib induces cell cycle arrestand autophagy and suppresses epithelial-to-mesenchymal transitioninvolving pi3k/akt/mtor and sirtuin 1-mediated signaling pathways inhuman pancreatic cancer cells,” Drug Des Devel Ther 2015, 9, 575-601;and Ruggeri, B.; Zhang, S. Y.; Caamano, J.; DiRado, M.; Flynn, S. D.;Klein-Szanto, A. J. “Human pancreatic carcinomas and cell lines revealfrequent and multiple alterations in the p53 and rb-1 tumor-suppressorgenes,” Oncogene 1992, 7, 1503-1511.

For OE33, see, Bamford, S.; Dawson, E.; Forbes, S.; Clements, J.;Pettett, R.; Dogan, A.; Flanagan, A.; Teague, J.; Futreal, P. A.;Stratton, M. R., et al., “The cosmic (catalogue of somatic mutations incancer) database and website,” Br J Cancer 2004, 91, 355-358 and Liu, D.S.; Read, M.; Cullinane, C.; Azar, W. J.; Fennell, C. M.; Montgomery, K.G.; Haupt, S.; Haupt, Y.; Wiman, K. G.; Duong, C. P., et al., “Apr-246potently inhibits tumour growth and overcomes chemoresistance inpreclinical models of oesophageal adenocarcinoma,” Gut 2015, 64,1506-1516.

TABLE 1 Status and expression of p53 in all cell types tested; wild-type(WT), mutant (MUT). P53 Cell Name Cell Type p53 Status Expression 769-PRenal adenocarcinoma WT Positive HCT-116 Colorectal carcinoma WTPositive SK-OV-3 Ovarian adenocarcinoma MUT/NULL Negative BxPC-3Pancreatic adenocarcinoma MUT Positive OE33 Esophageal adenocarcinomaMUT Positive

Ma et al. also used HCT-116 and surprisingly found only a slightreduction in viability whereas we found that viability was reduced to aslow as 3.6% (FIG. 6A). This is likely due to differences in plasmageneration, flow rate, and assay timing. The cell lines tested here alsoinclude both wild-type (WT) and mutant p53 (Table 1). Despite positivep53 expression, or status, the viability of these cell types wassignificantly affected by CAP treatment. However, the cell lines withwild-type p53 (769-P/FIGS. 5A and 5B, HCT-116/FIGS. 6A and 6B) tended torequire a lower dose of CAP to reduce viability compared to those withmutant p53 (BxPC-3/FIGS. 4A and 4B, OE33/FIGS. 9A and 9B).

Further experiments will investigate the effect of CAP on additionalcell lines as well as the potential of combined therapies. For celllines that require a higher dose of CAP to effectively reduce viability;that dose may be reduced with the addition of chemotherapy drugs.Combination therapy may also further reduce viability when combined withCAP. This would match the approach taken in a surgical setting and couldlead to improved patient outcomes.

Materials and Methods

Cold Plasma Device

Cold atmospheric plasma (CAP) was generated using a USMI SS-601 MCahigh-frequency electrosurgical generator integrated with a USMI ColdPlasma Conversion Unit and connected to a Canady Helios Cold PlasmaScalpel. Helium flow was set to a constant 1 L/min and 20P or 40P or 3L/min and power set to 40P, 60P, or 80P. The plasma scalpel was placedsuch that the tip of the scalpel was 1.5 cm (at 1 L/min) or 2 cm (at 3L/min) from the surface of the cell media and was not moved duringtreatment (FIGS. 10A and 10B).

Cell Culture

BxPC-3 pancreatic adenocarcinoma and 769-P renal adenocarcinoma werepurchased from ATCC (Manassas, Va.). OE33 esophageal adenocarcinoma waspurchased from Sigma-Aldrich (St. Louis, Mo.). HCT-116 colorectalcarcinoma and SK-OV-3 ovarian adenocarcinoma were generously donated byThe George Washington University. All cell lines were maintained withrequired culture media according to the supplier protocol. When cellsreached approximately 80% confluence, cells were seeded at aconcentration of 5×10³ or 10⁵ cells/well into 96-well or 12-well plates(USA Scientific, Ocala, Fla.), respectively, for cell viability assays.For BxPC-3×10⁴ cells were required for the 96-well assay.

Cell Viability Assay

Thiazolyl Blue Tetrazolium Bromide (MTT) assay was performed on thecells 48 hours after plasma treatment following the manufacturer'sprotocol. Briefly, cells were incubated with MTT at a concentration of0.5 mg/ml after 48 hours post treatment for 3 hours in a 37° C. and 5%CO₂ humidified incubator. Then, MT each well to dissolve the formazancrystals. All the MTT assay reagents were purchased from Sigma-Aldrich(St. Louis, Mo). The absorbance of the dissolved compound was measuredby BioTek Synergy HTX (Winooski, Vt.) microplate reader at 570 nm.

Statistics

All viability assays were repeated for at least 3 times with 2replicates each. Data was plotted by Microsoft Excel 2016 asmean±standard error of the mean. Student t-test or one-way analysis ofvariance (ANOVA) were used to check statistical significance whereapplicable. Differences were considered statistically significant for*p≤0.05.

The foregoing description of the preferred embodiment of the inventionhas been presented for purposes of illustration and description. It isnot intended to be exhaustive or to limit the invention to the preciseform disclosed, and modifications and variations are possible in lightof the above teachings or may be acquired from practice of theinvention. The embodiment was chosen and described in order to explainthe principles of the invention and its practical application to enableone skilled in the art to utilize the invention in various embodimentsas are suited to the particular use contemplated. It is intended thatthe scope of the invention be defined by the claims appended hereto, andtheir equivalents. The entirety of each of the aforementioned documentsis incorporated by reference herein.

What is claimed is:
 1. A system for performing cold atmospheric plasmatreatment on target tissue comprising: an RF energy source; a lowfrequency conversion unit; a gas control device configured to controlflow of a gas; a processor configured to control said RF energy source,said low frequency conversion unit and said gas control device to applyenergy from said low frequency conversion unit to gas flowing from saidgas control module to generate reactive species that attack cancer cellsbut do not damage healthy cells; a display; a graphical user interfacedisplayed on said display for controlling cold atmospheric plasmaprocedures; a data storage unit; and a database stored in said datastorage unit, said database comprising cell line identifying data andcold atmospheric plasma settings associated with the cell lineidentifying data; wherein, the graphical user interface has input meansfor a surgeon to enter cell line identifier data and in response to theentry of cell line identifier data the processor is configured toautomatically access the database in said data storage and adopt coldatmospheric plasma settings associated with the entered cell lineidentifier data in said database and display the adopted coldatmospheric plasma settings on the display.
 2. A system for performingcold atmospheric plasma treatment on target tissue according to claim 1,wherein the RF energy source is a monopolar electrosurgical generator.3. A system for performing cold atmospheric plasma treatment on targettissue according to claim 1, wherein the RF energy source, gas controldevice and conversion unit are modules within an integrated coldatmospheric plasma generator.
 4. A system for performing coldatmospheric plasma treatment on target tissue according to claim 3,wherein said processor is in said integrated cold atmospheric plasmagenerator.
 5. A system for performing cold atmospheric plasma treatmenton target tissue according to claim 4, wherein said data storage unit isin said integrated cold atmospheric plasma generator.
 6. A system forperforming cold atmospheric plasma treatment on target tissue accordingto claim 1, wherein said display comprises a touchscreen.
 7. A systemfor performing cold atmospheric plasma treatment on target tissueaccording to claim 6, wherein said touchscreen comprises a tabletcomputer.
 8. A system for performing cold atmospheric plasma treatmenton target tissue according to claim 3, wherein said data storage isexternal to said integrated cold atmospheric plasma generator.
 9. Asystem for performing cold atmospheric plasma treatment on target tissueaccording to claim 1, wherein said data storage comprises memory in saidprocessor.
 10. A system for performing cold atmospheric plasma treatmenton target tissue according to claim 1, wherein cold atmospheric plasmasettings comprise at least two of power, frequency, flow rate and time.11. An electrosurgical generator for performing cold atmospheric plasmatreatment on target tissue comprising: an RF energy module; a gascontrol module configured to control flow of a gas; a processorconfigured to control said RF energy module and said gas control moduleto apply energy from said RF energy module to gas flowing from said gascontrol module to generate reactive species that attack cancer cells butdo not damage healthy cells; a display; a graphical user interfacedisplayed on said display for controlling cold atmospheric plasmaprocedures; a data storage unit; and a database stored in said datastorage unit, said database comprising cell line identifying data andcold atmospheric plasma settings associated with the cell lineidentifying data; wherein, the graphical user interface has input meansconfigured to receive cell line identifier data and in response toreceipt of cell line identifier data the processor is configured toautomatically access the database in said data storage or memory andadopt cold atmospheric plasma settings associated with the entered cellline identifier data in said database and display the adopted coldatmospheric plasma settings on the display.